Nature is a prolific producer of small molecules that have evolved to interact with diverse biological targets. From a human health perspective, natural products have dramatically altered our lives by providing many front-line drugs as well as chemical probes to unravel basic molecular pathways germane to health and disease. Although natural products continue to provide about half of all new chemical entities approved as drugs by the US Food and Drug Administration, drug discovery during the latter part of the 20th century shifted away from natural products towards synthetic libraries. This paradigm shift reflected the complexity of small, natural libraries against the simplicity of large, combinatorial synthetic libraries and was rationalized in order to keep pace with the enormous capacity of industrial high-throughput screening programs. New drugs from combinatorial chemical libraries, however, did not materialize during this time period, while natural products continued as an important source. Natural products, like drugs, cover a chemical space that is much more diverse than combinatorial compounds, thereby reflecting the rich chemical diversity of this resource.
Recent technological advances in natural product research involving isolation, characterization, synthesis, and biosynthesis have rekindled an interest in their investigation in academia and industry. With the advent of modern molecular biology, the field of biosynthesis has blossomed over the past decade with new approaches to generate biosynthetic libraries that further extend natural product structural diversity into new chemical space. In vivo approaches involving combinatorial biosynthesis, mutasynthesis, and precursor-directed biosynthesis and complementary in vitro approaches that combine chemical synthesis and enzymology (chemoenzymatic synthesis) have led to impressive libraries of novel molecules never encountered in nature. Natural product structural classes that have been biosynthetically manipulated in this fashion include the polyketides, nonribosomal peptides, terpenoids, and alkaloids. Most progress in this burgeoning field has resided with the actinomycetes (soil bacteria), which offer impressive arrays of natural products whose biosynthetic genes are typically clustered and are thus readily amenable to genetic manipulation. One notable exception that is absent from the biosynthetic diversification platform, however, is the hybrid isoprenoid class of natural products.
Natural products, such as the isoprenoid (terpenoid) family of diverse chemical scaffolds have held significant interest for the synthetic organic chemistry community because they are both challenging synthetic projects and possess varied biological activities and medicinal properties. Within the terpenoid family, the total synthesis of sesquiterpene natural products and related analogs continue to dominate the chemical literature. The demand for a reliable production platform for structurally complex terpenes has increased dramatically over the last 10 years and is of growing interest. Elegant synthetic schemes for terpenoids have been developed, but suffer from low yields and low regio- and enantio-selectivity. Although engineered E. coli has the potential to make mg/L levels of sesquiterpene hydrocarbons, the more biologically active terpenes are highly functionalized with hydroxyl, methyl, acetyl, halide, carbohydrate, and peroxide functional groups that require multi-step biosynthetic mechanisms often tethered to endo-membrane systems conducive for metabolic coupling. By integrating biosynthetic complexity with synthetic diversification, it may be possible for many of these hurdles to the technological development of terpenoids to be overcome.
Moreover, hybrid compounds containing terpene-derived residues comprise a large and diverse group of natural products that command an important role in human health (see Table 1). Historically this class of compounds has provided important drugs (e.g., the anticancer agent vincristine, the antimalarial quinine and the immunosuppressant mycophenolate mofetil) as well as challenging synthetic targets (e.g., strychnine and reserpine). In addition to natural products, many important coenzymes (ubiquinone and plastoquinone) and vitamins (tocopherols, phylloquinones, and menaquinones), which function in electron transport systems, contain isoprenoid residues.
TABLE 1Representative hybrid isoprenoids, their sources and biological significanceNatural ProductSourceIsoprenoid HybridBiological Activitymycophenolic acidfunguspolyketideimmunosuppressantkhellinplantpolyketidebronchial asthmatetrahydrocannabinolplantpolyketidenarcotic, antiemeticrotenoneplantisoflavonoidinsecticidepsoralenplantcoumarinskin pigment and irritantnovobiocinbacteriumcoumarinantibioticlucidinplantquininemutagenemetineplanttetrahydroisoquinolinealkaloid emetic (ipecac)ergometrinefungusergotalkaloidoxytocicreserpineplantindolealkaloidantihypertensivevincristineplantindole alkaloidanticancerstrychnineplantindole alkaloidtoxinlyngbyatoxincyanobacteriumindole alkaloidinflammatory agentquinineplantquinoline alkaloidantimalarialcamptothecinplantquinoline alkaloidtopoisomerase/inhibitor
Nature has assembled a myriad of scaffolds to which isoprenoids have been attached, and these include polyketides (the so-called meroterpenoids), flavonoids, coumarins, quinones, alkaloids, phenazines, and the like. Often the terpenoid unit is further elaborated by electrophilic cyclization and oxidative chemistry upon attachment to its building block, thereby leading to the great structural diversity observed within this group. While most of these natural products contain a single isoprenoid unit of varying chain length, others harbor multiple isoprene units such as in the tetraprenylated benzoylphloroglucinol derivatives sampsoniones A-I.
The vast majority of hybrid isoprenoids are derived from eukaryotes, particularly plants. For instance, over a thousand monoterpenoid indole alkaloids have been characterized, making this a major class of plant alkaloids. On the other hand, terpenoids, and in particular hybrid isoprenoids, appear to have a limited distribution in prokaryotes. While actinomycetes are metabolically very rich bacteria and produce many important biosynthetic classes of natural products that include polyketides, nonribosomal peptides, aminoglycosides, and the like, the terpenoids are notably scarce. As a consequence, while other natural product structural classes have been biosynthetically exploited in the drug discovery arena, the hybrid isoprenoids are noticeably absent due to our limited understanding of their biosynthesis at the biochemical and genetic levels.
The majority of the basic understanding of how hybrid isoprenoids are biosynthesized in plants, fungi and bacteria is based on feeding experiments with labeled precursors. Enzymes and their encoding genes associated with interfacing isoprenyl diphosphates with their small molecule building blocks are very few and are mostly associated with plant natural products such as shikonin and with coenzymes and vitamins such as the ubiquinones, plastoquinones, menaquinones, and tocopherols. Very recently, two prokaryotic prenyltransferases (PTases) involved in the biosynthesis of the streptomycete antibiotics clorobiocin and novobiocin and the cyanobacterial toxin lyngbyatoxin were discovered. These soluble, monomeric PTases contrast with the membrane-associated PTases previously identified from eukaryotes.
Actinomycetes produce a limited set of pure and hybrid terpenoids. The antibiotic novobiocin was the first streptomycete natural product discovered with a terpenoid side chain; this group has since grown to include other members bearing naphthoquinones (naphterpin, furaquinocin, napyradiomycins), phenazines (lavanducyanin, aestivophoenin), shikimate-derived quinones, and other aromatic substrates (see FIG. 1B). Feeding experiments delineated a number of biosynthetic pathways, including those to novobiocin, naphterpin, and furaquinocin, and revealed that actinomycetes utilize both the mevalonate and nonmevalonate (methyl-D-erythritol 4-phosphate (MEP)) pathways to synthesize their isoprene building blocks.
The development of novel methodologies related to natural products chemistry and biosynthesis is of growing interest. Prenylated aromatic natural products appear to be a very promising class of therapeutically compounds. The prenylation of aromatic compounds often leads to significant alteration in the bioactivity profile of a compound, by both the creation of a novel C—C bond and also the introduction of one or more double bonds in the framework of the final product. Such compounds can affect a wide variety of biological systems in mammals and include roles as antioxidants, anti-inflammatories, anti-virals, anti-proliferatives, and anti-cancers.
Prenyltransferases (PTases) are ubiquituous enzymes that catalyze the alkylation of electron rich prenyl acceptors by the alkyl moieties of allylic isoprene diphosphates. Prenyltransferases utilize isoprenoid diphosphates as substrates, and catalyze the addition of the acyclic prenyl moiety to isopentenyl diphosphate (IPP), higher order prenyl diphosphates, aromatic rich molecules and proteins. Until now, only a few “aromatic” prenyltransferases have been isolated, each of which has been shown to interact with only a limited range of substrate(s) and/or prenyl donors. Such prenyltransferases have otherwise only been nominally characterized; and none of such prenyltransferases have been characterized at the structural level.
Accordingly, there is a need in the art for the identification of novel enzymes capable of promoting the prenylation of aromatic compounds, as well as compounds which can modulate the prenylation of aromatic compounds. These and other needs are addressed by the present invention, as described in greater detail in the specification and claims which follow.